ES2365077T3 - ANTI-PENIC ANTIGEN PEPTIDE DERIVED FROM SPARC AND PHARMACEUTICAL COMPOSITION THAT INCLUDES THE SAME. - Google Patents

Abstract

A peptide of any of the following: (A) a peptide consisting of the amino acid sequence shown in any of the SEC. ID. no 1 to no 3; or (B) a peptide consisting of an amino acid sequence comprising a substitution or addition of one or two amino acids with respect to the peptide consisting of the amino acid sequence shown in any of the SEC. ID. No 1 to 3, and it has the capacity to produce cytotoxic T lymphocytes (killers); or (C) a peptide consisting of an amino acid sequence comprising an addition of one to 10 amino acids with respect to the peptide consisting of the amino acid sequence shown in any of the SEC. ID. No 1 to 3, and it has the capacity to produce cytotoxic T lymphocytes (killers).

Description

Technical field

The present invention relates to new peptides that are effective as a vaccine against cancers that express SPARC to a large extent, such as stomach cancer, pancreatic cancer or malignant melanoma (melanoma), and medicaments comprising the aforementioned peptides used for the treatment and / or prevention of tumors.

State of the art

Compared to western countries, the morbidity of stomach cancer is high in Asian countries such as Japan and China. As a result of the dissemination of medical examinations, the widespread use of digestive endoscopes, and the development of inspection techniques, it has become possible to detect stomach cancer at an early stage and, therefore, the number of patients suffering from This type of cancer has been decreasing. However, stomach cancer remains the second leading cause of death due to malignant neoplasia in the Japanese population. Therefore, stomach cancer is still the leading cause of death. Among the various types of stomach cancers, diffuse stomach cancer (escirro) occurs in young people compared to another type of stomach cancer (adenocarcinoma). Said diffuse stomach cancer (escirro) tends to progress rapidly, and distant metastasis or peritoneal metastasis occurs frequently, resulting in a poor prognosis. In many cases of escirro stomach cancer, it has already become impossible to carry out surgical removal at the time of diagnosis. Even if it is still possible to remove the tumor, cancer often comes back after treatment. Consequently, it is very desirable to introduce a new method of treatment.

The death toll from pancreatic cancer tends to increase in Japan. 21,148 people died due to pancreatic cancer in 2003. Currently, said pancreatic cancer constitutes 6.8% of cancers that cause death in Japan. That is, pancreatic cancer occupies the fifth cause of death after lung cancer, stomach cancer, colon cancer and liver cancer. Demographic data in the world were analyzed, and the age-adjusted mortality rate, which is used for comparison between the mortality rates of populations with different age structures, was calculated. As a result, in 2000, in the case of 100,000 men, 8.6 people died in Japan due to pancreatic cancer, while 7.3 people died in the United States and 6.3 to 7.0 people died in the United Kingdom due to the same type of cancer. In the case of every 100,000 women, 4.9 people died due to pancreatic cancer in Japan, while 5.3 people died in the United States and 4.8 to 5.1 people died in the United Kingdom due to the same type Of cancer. Thus, the death rate from pancreatic cancer in Japan has become at the same level as those in Western countries.

Given the age-adjusted rate in 2000 (100,000 people of the world population), in the case of men, 8.6 people died due to pancreatic cancer in Japan, while 7.3 people died in the States United and 6.3 to 7.0 people died in the United Kingdom due to the same type of cancer. In addition, in the case of women, 4.9 people died due to pancreatic cancer in Japan, while 5.3 people died in the United States and 4.8 to 5.1 people died in the United Kingdom due to to the same type of cancer. Thus, the death rate from pancreatic cancer in Japan has reached the same level as those in Western countries. Despite the development of diagnostic imaging, at present, approximately 40% of all Japanese patients with pancreatic cancer suffer from progressive pancreatic cancer that involves distant metastases, and in addition, there are also many cases where the cancer, after reaching a stage of locally advanced cancer, in which tumor cannot be removed. The 5-year relative survival rate of all patients with pancreatic cancer is 4.3% in cases of diagnosis in 1996. Although this rate tends to be higher than the conventional survival rate (2% to 3 %), remains low. As for the factors of developing pancreatic cancer, it has been suggested that various factors such as lifestyle as smoking, adiposis, food, drink alcohol and coffee, as well as chronic pancreatitis, diabetes, genetic factors, etc. They are involved in the onset of pancreatic cancer.

Pancreatic cancer has no specific symptoms, and therefore, in many cases, the cancer has already progressed when certain symptoms appear. As a result, the 5-year survival rate of all patients is 5% or less, and the prognosis after diagnosis is extremely low. Due to the difficulty in the diagnosis of pancreatic cancer, the rate of this cancer as a disease causing death from cancer gradually increases, particularly in advanced countries. At present, multidisciplinary therapy has been carried out that includes surgical removal as the main treatment, radiotherapy, and chemotherapy. However, no drastic improvement in therapeutic effects can be obtained, and therefore, the introduction of a new therapeutic strategy is urgently necessary.

Melanoma is a type of skin cancer, which is often called malignant melanoma. Among the various types of skin cancers, melanoma is highly likely to become infiltrating and metastatic and has the highest degree of malignancy, so it is very feared. Among the cells that make up the skin, the cells generate the melanin pigment. These cells are called melanocytes. When these melanocytes become cancerous, melanoma occurs. In addition, the frequency of occurrence of melanoma has been increasing, especially among Caucasians, as a result of increased exposure to ultraviolet rays, due to a reduction in the ozone layer in the atmosphere caused by the destruction of the environment. ambient.

In Japan, the number of melanoma cases varies from 1.5 to 2 people per 100,000 in the general population. Therefore, it is estimated that approximately 1,500 to 2,000 people develop melanoma a year. On the other hand, in Western countries, more than a dozen people develop melanoma for every 100,000 in the general population. In particular, in Australia, twenty or more people develop melanoma, for every 100,000 in the general population, and therefore it is known that the number of cases of melanoma in Australia is the highest in the world. In such circumstances, people living in Europe, the United States and Australia are interested in melanoma, and pay attention to the onset of melanoma. In addition, surprisingly, the onset of melanoma tends to be increasing year after year in Japan, as well as abroad. According to recent studies, the number of deaths from melanoma is approximately 450 in Japan. Melanoma develops regardless of age. However, the number of cases of this disease increases for those over 40, and is the highest for people aged 60 to 70 years and 70 to 80 years. The appearance of this disease in childhood is extremely rare, but this does not mean that the disease never develops in childhood. Recently, the onset of melanoma tends to be increasing in young patients in their 20s and 30s. Melanoma develops regardless of sex, and both male and female patients suffer from this disease. In the case of Japanese patients, the site in which melanoma is the most likely to develop is in the sole (the sole of the foot), and accounts for 30% of all cases of melanoma. As characteristics of Japanese patients, melanoma also develops in the feet and parts of the fingernails. In addition, as in the case of Western patients, melanoma develops in all parts of the skin, such as the body, hands, feet, face and head.

Currently, methods that can be applied to treat melanoma include surgical treatment, chemotherapy, and radiation therapy. However, as a therapy to relieve the symptoms of metastatic cancer or intractable cancer, to which the previous therapy cannot be applied, an immunotherapy to improve the immunity of a cancer patient to suppress cancer growth has become A focus of attention. This type of immunotherapy is really effective for some patients.

On the other hand, with the development of molecular biology and tumor immunology in recent years, it has been shown that cytotoxic T lymphocytes (killers) and helper T lymphocytes recognize the peptides generated by the degradation of proteins very and specifically expressed in cancer cells, which are presented on the surface of cancer cells or antigen presenting cells through HLA molecules, and that have an immune reaction to destroy said cancer cells. On the other hand, a large number of tumor antigenic proteins and peptides derived from them, which stimulate said immune reaction to attack cancers, have been identified, and the clinical application of a tumor-specific immunotherapy for the antigen has been advanced.

HLA class I molecules are expressed on the surfaces of all nucleated cells in a body. The proteins generated in the cytoplasms and nuclei provide peptides generated as a result of degrading in the cells, and which are expressed on the surface of these cells. On normal cell surfaces, peptides derived from normal autologous proteins bind to HLA class I molecules, and T lymphocytes from the immune system neither recognize nor destroy such peptides bound to class HLA molecules.

I. On the other hand, in a process in which cancer cells become a cancer, said cancer cells can express large amounts of proteins, which are barely expressed or are exposed only in small amounts in normal cells. If a peptide generated by the degradation in the cytoplasm of said protein that is highly expressed specifically in a cancer cell binds to a HLA class I molecule and is expressed on the surface of said cancer cell, a killer T lymphocyte recognizes the peptide and destroy only the cancer cell. Furthermore, by immunizing said cancer-specific antigen or peptide for a natural person, it is possible to destroy the cancer cells and suppress the growth of a cancer, without affecting the normal cells. This is known as cancer immunotherapy using a cancer-specific antigen. On the other hand, a class II HLA molecule is expressed primarily on the surface of an antigen presenting cell. Said class II HLA molecule binds to peptides derived from a cancer-specific antigen generated by incorporating the cancer-specific antigen from outside the cell and degrading it into the cell, and is expressed on the surface of the cell. A collaborative T lymphocyte, which has recognized the peptides bound to the HLA class II molecule, is activated to generate various cytokines that activate other immunocompetent cells, in order to provoke or strengthen an immune reaction against a tumor.

Thus, if an immunotherapy directed at an antigen specifically expressed at a high level in said cancer can develop, it can provide a therapeutic method effectively eliminating the cancer alone, without altering normal autologous organs. On the other hand, it is envisioned that such immunotherapy can provide a therapeutic method applicable to patients suffering from cancer in the terminal stage, for which no other treatment can be applied. In addition, if a cancer-specific antigen and the peptide are administered as a vaccine to a human being with a high risk of developing a cancer, there is a possibility that the onset of cancer can be prevented.

First, the present inventors have performed an analysis of gene expression throughout the genome. 23,040 types of genes have been analyzed in stomach cancer tissues and in normal tissues that use a cDNA chip. As a result, in 11 of the 20 cases of patients with diffuse infiltrating stomach cancer, the inventors have identified segregated acidic and cysteine-rich protein (SPARC), which is a very expressed gene in stomach cancer tissues, and whose level of expression is 5 or more times greater than that of normal tissues (130,000 times higher on average) (Figure 1). The SPARC gene is expressed at a low level also in normal adipose tissues, the mammary gland, the ovary, the spinal cord, the testicles, the uterus, the placenta, etc. However, the level of expression of the SPARC gene in any of the organs mentioned above is lower than normal normal gastric mucous membrane by a factor of 5 times or less (Figure 2).

Other researchers had already reported that SPARC is not only very expressed in diffuse infiltrating stomach cancer, but also expressed in pancreatic cancer and melanoma. On the other hand, the present inventors have discovered that SPARC is secreted in the serum of patients with melanoma, and that SPARC may be a tumor marker useful in particular for the early detection of melanoma (Japanese patent application number 2004-303688, and Clinical Cancer Research 11: 8079-8088, 2005).

SPARC is a 43 kDa secretory acid protein consisting of 286 amino acids. This protein is rich in cysteine, and travels to the nucleus during the cell division phase. In addition, SPARC controls the interaction between an extracellular matrix protein and a cell, so that it can also be associated with cell growth control. Since SPARC is expressed in osteoblasts, thrombocytes and wound areas, this protein is considered to be associated with tissue repair and reconstruction. On the other hand, it has also published that SPARC also expresses a lot in cancers such as melanoma or osteosarcoma and in interstitial cells of tumors, and that SPARC expression correlates with the prognosis, infiltration or metastasis of tumors .

[Non-patent document 1] Ikuta Y. et al. ,, Clinical Cancer Research 11: 8079-8088, 2005. [Patent document 1] Japanese patent application No. 2004-303688

Description of the invention

Problems to be solved by the invention

An objective of the present invention is to develop a method to improve the immunity of a patient with metastatic cancer or intractable cancer, for which surgical therapy, chemotherapy and radiotherapy are used as treatments for tumors that largely express SPARC, such as diffuse infiltrating stomach, pancreatic cancer or melanoma, can hardly be applied, in order to relieve the symptoms of such cancers and in order to carry out an immunotherapy suppressing the growth of said cancers. That is, an objective of the present invention is to identify a peptide derived from a SPARC protein that is overexpressed in the cancerous tissue, which is capable of provoking a potent immune response to the cancers mentioned above without producing harmful phenomena to cancer patients. , and apply the identified peptide to a tumor immunotherapy. That is, an objective of the present invention is to identify a peptide derived from the SPARC protein that can produce killer T lymphocytes and human helper T lymphocytes with reactivity to tumors, and provide a means to carry out a tumor immunotherapy in patients with Several types of cancers that largely express SPARC.

Means to solve the problems

The present inventors have identified a gene, SPARC, which is highly expressed in diffuse infiltrating stomach cancer, by performing DNA chip analysis in the aforementioned stomach cancer and various normal tissues. SPARC expression is also observed in several types of normal tissues. However, the level of SPARC expression in normal tissues is significantly lower than in cancerous tissues. In order to examine the presence or absence of antitumor immunity induction by SPARC-specific killing T lymphocytes, Balb / c mice expressing Kd mouse molecules were used, whose amino acid sequence characteristics of the bound peptide are identical to those of HLA-A24 which is the most frequent HLA class allele in the Japanese population. Human SPARC possesses 95% of the homologous amino acid sequence compared to the SPARC mouse. In this way the peptides that consist of amino acid sequences shared between humans and mice, and that have a motif of joint shared by humans HLA-A24 and the mouse Kd, were synthesized. Then, BALB / c (expressed Kd) mice were immunized with dendrocytes from the bone marrow, in which a mixture of peptides had been loaded, and the inventors investigated whether killer T lymphocytes reactive with cancer cells expressing SPARC are produced or no. On the other hand, in relation to mice that had previously been treated by the same immunization method as described above, the inventors investigated whether the growth of transplanted cancer cells expressing mouse SPARC is suppressed and the survival time of the Mice is prolonged or not. In addition, it was also examined whether or not a harmful phenomenon appears along with the appearance of an autoimmune phenomenon in mice immunized with peptide. As a result, it was found that the peptide having the amino acid sequence shown in any of the SEQ. ID. No. 1 to 3 is capable of producing killer T lymphocytes that destroy cancer cells expressing SPARC. On the other hand, the growth of mouse transplanted cancer cells expressing SPARC is suppressed in mice immunized with the mentioned peptide and therefore the survival time of the mice is prolonged. The present invention has been carried out on the basis of these results.

The present invention provides the following invention.

(one)

A peptide from any of the following:

(TO)

a peptide consisting of the amino acid sequence shown in any of the SEC. ID. No. 1 to No. 3; or

(B)

a peptide consisting of an amino acid sequence comprising a substitution or addition of one or two amino acids with respect to the peptide consisting of the amino acid sequence shown in any of the SEC. ID. No. 1 to 3, and which has the capacity to produce cytotoxic T lymphocytes (killers).

(2)

An immunological agent for cancers, comprising at least one type of peptide of (1).

(3)

A medicament for the treatment and / or prevention of tumors, comprising at least one type of peptide of (1).

(4)

A peptide for the induction of antigen presenting cells that has a great capacity to induce T-lymphocytes reactive with the tumor, comprising at least one type of peptide of (1).

(5)

A peptide for the induction of T lymphocytes reactive with the tumor, comprising at least one type of peptide of (1).

(6)

An agent for the induction of antigen presenting cells that has a great capacity to induce tumor-reactive T lymphocytes, which comprises a gene that codes for a peptide of any of the following:

(TO)

a peptide consisting of the amino acid sequence shown in any of SEC. ID. No. 1 to 3, or

(B)

a peptide consisting of an amino acid sequence comprising a substitution or addition of one or two amino acids with respect to the peptide consisting of the amino acid sequence shown in any of the SEC. ID. No. 1 to No. 3, and which has the capacity to induce killer T lymphocytes.

(7)

A killer T lymphocyte, a helper T lymphocyte, or an immunocyte population comprising said cells, which is induced using the peptide of (1).

(8)

An antigen presenting cell, which has a complex of an HLA molecule and the peptide of (1).

(9)

The antigen presenting cell of (9), which is induced using the agent of (4) or (6).

Best way to carry out the invention

(1) Peptide of the present invention, and the immune agent for cancers comprising the same

The peptide of the invention is described below:

(A), a peptide consisting of the amino acid sequence shown in any of the SEC. ID. No. 1 to 3, or

(B) a peptide consisting of an amino acid sequence comprising the substitution or addition of one or more amino acids with respect to the amino acid sequence shown in any of the SEC. ID. No. 1 to 3, and which has the capacity to induce killer T lymphocytes.

Expression of a peptide with activity to stimulate killer T lymphocytes reactive with the tumor.

A process for obtaining / producing the peptide of the invention is not particularly limited. A chemically synthesized peptide, or a recombinant peptide produced by genetic recombination can be used.

When a chemically synthesized peptide is obtained, the peptide of the invention can be synthesized by a chemical synthesis method such as an Fmoc method (fluorenylmethyloxycarbonyl method) or a tBoc method (t-butyloxycarbonyl method), for example. In addition, the peptide of the invention can also be synthesized using various types of commercially available peptide synthesizers.

When the peptide of the invention is produced in the form of a recombinant protein, DNA with a nucleotide sequence encoding the aforementioned peptide, a mutant thereof or a homologue thereof is obtained, and then introduced into a preferred expression system. , to produce the peptide of the present invention.

As an expression vector, a vector capable of replicating autonomously in a host cell or capable of being incorporated into the chromosome of a host cell can preferably be used. An expression vector comprising an activator in a position capable of expressing a gene encoding the peptide is used. In addition, a transformant having a gene encoding the peptide of the present invention can be produced by introducing the aforementioned expression vector into a host. As a host, anyone between a bacterium, yeast, an animal cell and an insect cell can be used. An expression vector can be introduced into a host according to a known method, depending on the type of said host.

In the present invention, the transformant produced above is cultured, and the peptide of the invention is then generated and accumulated in a culture. Then, the peptide of the invention is collected from the culture, in order to isolate a recombinant peptide.

When said transformant is a prokaryotic such as Escherichia coli or a eukaryotic such as yeast, a medium used for the cultivation of such microorganisms, it can be a natural medium or a synthetic medium, as long as it contains a carbon source, a source of nitrogen, inorganic salts and the like that can be assimilated by the aforementioned microorganisms, and is capable of effectively carrying out the culture of the transformant. On the other hand, said culture can be carried out under conditions that are usually applied for the cultivation of the microorganisms mentioned above. Once the culture is finished, the peptide of the present invention can be isolated and purified from the culture of the transformant according to a usual method of isolation and purification of peptides.

The term "one or more amino acids" is used herein in the sense generally of 1 to 10 amino acids, preferably 1 to 8 amino acids, more preferably 1 to 5 amino acids, and particularly preferably 1 to 3 amino acids (per example, 1, 2 or 3 amino acids).

A peptide consisting of an amino acid sequence comprising a substitution or addition of one or more amino acids with respect to the peptide consisting of the amino acid sequence shown in any of the SEC. ID. No. 1 to No. 3 may be properly produced or acquired by persons skilled in the art based on information on the amino acid sequence shown in any of the SEC. ID. nº 1 to nº

3. That is, a peptide consisting of an amino acid sequence comprising a substitution or addition of one or more amino acids with respect to the amino acid sequence shown in any of SEC. ID. No. 1 to No. 3, and which has the capacity to induce cytotoxic T lymphocytes, can be produced by any method known to persons skilled in the art, such as the chemical synthesis mentioned above, genetic engineering means or mutagenesis. For example, directed mutagenesis, which is a means of genetic engineering is useful because it is a means for the introduction of a specific mutation in a specific position. Such site-directed mutagenesis can be performed by a method described in the Molecular Cloning: A laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989 (hereinafter abbreviated as Molecular Cloning 2nd ed.), Current Protocols in Molecular Biology, Supplement 1 to 38, John Wiley & Sons (1987-1997) (hereinafter abbreviated as Current Protocols in Molecular Biology), etc.

As described later in the examples, the aforementioned peptide of the present invention is capable of inducing immunity against cancer. Therefore, the present invention provides an immunological agent for cancers, which comprises the peptide of the present invention.

The immunological agent of the present invention used for cancers is used in vitro, ex vivo or in vivo, and preferably ex vivo, so that it can induce killer T lymphocytes, helper T lymphocytes, or a population of immunocytes comprising said cells, thus imparting immunity against cancer.

(3) Slayer T lymphocytes, helper T lymphocytes, or a population of immunocytes comprising said cells

The present invention also relates to killer T lymphocytes, helper T lymphocytes or a population of immunocytes comprising said cells, which is induced by in vitro stimulation using the peptide of the present invention. For example, when peripheral blood lymphocytes or infiltrating lymphocytes in the tumor are stimulated in vitro with the peptide of the present invention, activated T lymphocytes that react in the tumor are induced. Therefore activated T lymphocytes can be used effectively for adoptive cancer immunotherapy. In addition, the peptide of the present invention is allowed to be expressed in dendrocytes that are strong antigen presenting cells in vivo or in vitro, and dendrocytes expressing the arc antigenic peptide are then administered to elicit an immune response to tumors.

Preferably, a killer T lymphocyte, a helper T lymphocyte or a population of immunocytes comprising said cells can be induced by ex vivo or in vitro stimulation using the peptide of the present invention and an immunostimulant. Examples of said immunostimulant used herein include a cell growth factor and a cytokine.

The killer T lymphocyte, the collaborative T lymphocyte, or of the population of immunocytes thus obtained comprising said cells is transferred to a body, so that the tumor can be suppressed and the cancer can be prevented and / or treated.

In addition, using the peptide of the present invention, a killer T lymphocyte, a helper T lymphocyte or a population of immunocytes comprising said cells can be produced, which is capable of suppressing tumor growth as described above. Accordingly, the present invention provides a cell culture solution comprising reactive T lymphocytes in the tumor and the peptide of the present invention. Using said cell culture solution, killer T lymphocytes, helper T lymphocytes or a population of immunocytes comprising said cells can be produced, which is capable of suppressing tumor growth. Furthermore, the present invention also provides a cell culture kit for producing killer T lymphocytes, helper T lymphocytes or a population of immunocytes comprising said cells, comprising the cell culture solution mentioned above and a cell culture vessel.

(4) Medication of the present invention for treatment and / or prevention of tumors (cancer vaccine)

Since the peptide of the invention is capable of inducing specific killer T cells from cancer cells, it can be expected as an agent for the treatment and / or prevention of cancer. For example, bacteria such as BCG (Bacillus Calmette-Guérin), which was transformed with recombinant DNA produced by the incorporation of a gene encoding the peptide of the invention into a suitable vector, or virus such as the virus of the vaccine, in whose genome the DNA encoding the peptide of the present invention has been incorporated, can be used effectively as a live vaccine for the treatment and / or prevention of human cancers. It should be noted that the dose and method of administration of a cancer vaccine are the same as in the case of ordinary smallpox vaccine or BCG vaccination.

That is, the DNA encoding the peptide of the invention (which is used as is, or is in the form of plasmid DNA incorporated into an expression vector), or a recombinant virus or a recombinant bacterium comprising the DNA mentioned above, is it can be administered as a vaccine against cancer to mammals including a human being, directly or in a state in which it is dispersed in an adjuvant. Similarly, the peptide of the invention can also be administered as a cancer vaccine in a state in which it is dispersed in an adjuvant.

Examples of an adjuvant used in the present invention include an incomplete Freund's adjuvant, BCG, trehalose dimicolate (TDM), lipopolysaccharide (LPS), an alum adjuvant and a silica adjuvant. From the standpoint of the ability to induce antibodies, an incomplete Freund's adjuvant (AIF) is preferably used.

The type of cancer is not particularly restricted herein. Specific examples of cancer include stomach cancer, colon cancer, esophageal cancer, pancreatic cancer, liver cancer, gallbladder cancer, cholangiocarcinoma, lung cancer, breast cancer, thyroid cancer, melanoma (malignant melanoma), cancer of skin, osteosarcoma, pheochromocytoma, head and neck cancer, brain tumor, chronic myelogenous leukemia, acute myelogenous leukemia, malignant lymphoma, kidney cancer, bladder cancer, prostate cancer, testicular cancer, uterine cancer, ovarian cancer and soft tissue sarcoma. Of these, cancers that express a lot of SPARC, such as stomach cancer (particularly diffuse infiltrating stomach cancer), pancreatic cancer and melanoma (malignant melanoma), are typical examples.

The peptide of the present invention acts as an epitope of T lymphocyte to induce specific killer T cells of cancer cells or helper T cells. Thus, the peptide of the present invention is useful as an agent for the prevention and / or treatment of human cancers. As a real use, the peptide or antibody of the present invention can be administered as an injectable product, directly or together with a pharmaceutically acceptable carrier and / or diluent, and when necessary, also together with the auxiliary substances mentioned below. On the other hand, the peptide or antibody of the present invention can also be administered by a method such as atomization, by transdermal absorption through the mucosa. The term "vehicle" is used here in the sense of human serum albumin, for example. In addition, as a diluent, PBS, distilled water or the like can be used.

As a dose, the peptide of the present invention can be administered in the range between 0.01 and 100 mg per adult and per administration. However, the dose is not limited to the aforementioned range. The pharmaceutical form is not particularly limited, either. A lyophilized product, or a granule produced by adding an excipient such as sugar, may also be available.

Examples of an auxiliary substance, which can be added to the agent of the invention to increase the inductive activity of T-reactive lymphocytes in the tumor, include: muramyl dipeptide (MDP); bacterial components, such as BCG bacteria; ISCOM described in Nature, vol. 344, p. 873 (1990); Saponin QS-21 described in J. Immunol. vol. 148, p. 1438 (1992); liposomes; and aluminum oxide. In addition, they can also be used as immunostimulatory auxiliary substances, such as lenthinan, schizophyllan or Picibanil. Other examples of products used herein as auxiliary substances include: cytokines to increase the growth or differentiation of T lymphocytes, such as IL-2, IL-4, IL-12, IL-1, IL-6 or FNT ; α galactosylceramide to activate NKT cells; CpG that binds to a Toll-like receptor to activate the natural immune system; and lipopolysaccharide (LPS).

On the other hand, the aforementioned antigen peptide is added in vitro to cells obtained from an HLA-A24 positive patient or allogeneic cells isolated from any other person who is HLA-A24 positive, followed by the presentation of cell antigen . Then, the cells are administered in the patient's blood vessel, so that killer T lymphocytes can be effectively induced in the patient's body. In addition, the

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The present peptide is added to the peripheral blood lymphocytes of a patient, and the mixture obtained is then cultured in vitro. Thus, killer T lymphocytes can be induced in vitro, and can then be returned to the patient's blood vessel. Such therapy that includes cell transfer has already been carried out as a method for the treatment of cancers, and so So much is a method well known to people skilled in the art.

By introducing the peptide of the invention into a body, the reactive T lymphocytes in the tumor are induced, and, consequently, an antitumor effect can be anticipated. On the other hand, when lymphocytes are stimulated by the peptide of the present invention ex vivo or in vitro present, activated T lymphocytes are induced. Activated T lymphocytes are injected into the affected area. Therefore, this technique can be used effectively for adoptive immunotherapy.

The present invention will be described in more detail in the following examples.

Examples

Example 1

(1) Analysis of cDNA chips

Regarding the cancerous tissues removed from a patient with diffuse infiltrating stomach cancer, a cancerous tissue was distinguished from non-cancerous tissues by laser microdissection, and both the cancerous and non-cancerous parts were then cut. Subsequently, RNA was extracted from each tissue. When cDNA was synthesized from said RNA by a reverse transcription reaction, cDNA generated from cancerous tissue was fluorescently labeled with Cy5, and cDNA generated from noncancerous tissue was fluorescently labeled with Cy3. Subsequently, the two cDNA preparations were mixed to obtain target DNA. Hybridization was carried out on a glass slide in which the probe cDNA has been deployed, and the non-specific junctions were then removed by washing. Then, the fluorescence images obtained after hybridization were captured with a CCD camera or a fluorescence scanner, and then displayed with false colors (Cy5: red; Cy3: green). At the same time, the relationship between the two types of fluorescence intensity (R / G) was calculated, and indicated as a gene expression profile. On the other hand, the expression of 23,040 types of genes in the cancerous and non-cancerous parts of 20 patients with diffuse infiltrating stomach cancer was subjected to comparative research, and 15 types of genes whose expression ratio in the cancerous tissue / non-cancerous tissue Cancerous was 5 or more were selected (Figure 1).

Subsequently, the expression profile of the 23,040 types of genes in normal tissues of 29 organs (including the four embryonic organs) and the genes whose expression level was low in these normal organs were selected were analyzed. In the case of SPARC, which the authors selected as a tumor antigen in this experiment, the expression ratio of cancerous / non-cancerous portions was 5 or higher (133,359 times on average) in 11 of every 20 patients with infiltrating stomach cancer diffuse. SPARC was expressed in some normal tissues, but the levels of expression in these non-cancerous tissues were significantly lower than in the cancerous tissues (Figure 2).

Example 2

(2) Selection of repertoires of SPARC peptide restricted to human HLA-A24 and mouse Kd

In human and mouse SPARC that has a 95% homology, the following peptides were selected from the SPARC fragment whose amino acid sequences were shared between humans and mice. All of the amino acid sequences of the SPARC molecules were investigated based on known information about amino acid sequences frequently observed in peptides that have strong binding affinity for both human HLA-A24 and mouse Kd molecules. The repertoires of peptides that were supposed to have high binding affinity for the two types of molecules were selected using a computer program. As a result, 4 types of peptides were selected as candidate peptides (Table 1).

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Table 1. SPARC derived peptides that possibly have binding affinity for both human HLA-A24 and mouse Kd molecules

Peptide

Sequence Initial position Link Score

A24

Kd

SPARC-Kd-1 SPARC-Kd-2 SPARC-Kd-3 SPARC-Kd-4

DYIGPCKYI HFFATKCTL EFPLRMRDWL MYIFPVHWQF 143 123 161 125 75 20 30 210 400 1382 960 120

Provided by http; // bimas, dcrt.nih.gov / cgi-bin / molbio / kon parker comboform SPARC-Kd-1: SEC. ID. No. 1 SPARC-Kd-3: SEC. ID. No. 2 SPARC-Kd-4: SEC. ID. nº 3

(3) Dendrocyte Vaccine (DC)

Dendrocytes from bone marrow (BM-DC) were produced in bone marrow cells from BALBc bone marrow using GM-CSF as described previously by the authors (Nakatsura, T. et al., Clin. Cancer Res. 10 , 8630-8640, 2004). Thus, the BM-DC obtained were cultured with a mixture of the 4 types of peptides selected as described above at a concentration of 10 mM for 2 hours, and the BMDC (5 × 105 cells) pulsed with peptide were then administered, intraperitoneally to each mouse. After an interval of 1 week, the same peptide-pressed BM-DCs were administered to the mouse twice, so that the mouse was strongly immunized. Next, the splenocytes of the mice were recovered and the induction of killer T lymphocytes was evaluated. In order to strictly analyze the induction of killer T lymphocytes from CD8 + T lymphocytes, after spleen collection, CD4 + T lymphocytes were removed from the collected splenocytes, using MACS grains, and the remaining cells were used for analysis.

The following protocols were used for the generation and characterization of SPARC-specific killing T lymphocytes (the day of recovery of splenocytes from the immunized mouse was defined as day 0).

Day 21 (1) GM-CSF was added to the bone marrow cells of a BALB / c mouse, in order to initiate the induction of dendrocytes from the bone marrow (hereinafter referred to as BM-DC).

Day-14 (2) A mixture of the 4 types of SPARC peptides was added to the induced BM-DC, and 2 hours later, 5 × 105 cells were administered intraperitoneally to each mouse.

Steps (1) and (2) were repeated twice every two weeks.

Day 0 The splenocytes of the immunized BALB / c mouse were recovered. The cells were cultured together with BM-DC previously pulsed with each SPARC peptide for 2 hours. Then, the resulting cells were grown together for 6 days.

Day 6 In order to detect the killer T lymphocytes that specifically recognize the SPARC peptides, a Cr release assay was carried out. T2Kd cells, a male RL cell line 1, a Meth A cell line and a cell line were selected. Balb / 3T3 as target tumor cells for killer T lymphocytes.

(4) Analysis of the cytotoxic effects of SPARC-specific killing T lymphocytes on tumor cells by Cr release assay

The cytotoxic activity of those in killer T lymphocytes specific for induced SPARC was analyzed as follows. The T2Kd cell is a cell line obtained by introducing a Kd gene into a mouse T2 cell line that lacks the expression of a TAP gene. Only in one case where a peptide added from outside is bound to an MHC class I molecule, due to insufficient TAP, the expression of a complex of the MHC class I molecule and the peptide is stably expressed in the cell surface A male RL cell line 1 is T-cell leukemic cells from BALB-c mice and do not express SPARC. The two types of cells above do not express SPARC. On the other hand, a Meth A cell line and a BALB / 3T3 cell line are the fibrosarcoma cell line from the BALB / c mouse and the fibroblast cell line, respectively. These two cell lines express SPARC. The aforementioned cell lines were labeled with radioactive chromium (Cr), and then cultured with the killer T lymphocytes mentioned above for 4 hours. Next, a culture supernatant was recovered, and the amount of radioactive Cr released from dead cells was quantified, whereby cytotoxic activity was detected.

As a result, no cytotoxicity was observed to T2Kd cells or RL cells in male cells 1, which did not express SPARC. However, cytotoxicity was specifically observed to T2Kd lymphocytes, which expressed SPARC peptides in the context of the surface Kd molecule by the loading of SPARC Kd peptides 1, 3 and 4, and also to Meth A and BALB / 3T3 , which spontaneously expressed SPARC (Figure 3).

Example 3

(5) Induction of antitumor immunity in BALB / C mice by immunization with SPARC peptide

(Process)

BM-DC was cultured with a mixture of 1, 3 and 4 Kd SPARC peptides (10 µM for each) for 2 hours. Then, 5 x 105 cells were administered intraperitoneally to each mouse. After an interval of one week, a BM-DC loaded with peptide was administered to the mice in the same manner, so that the mouse was immunized twice in total. Seven days later, a mouse fibrosarcoma cell line, Meth A (1 x 106 cells), which is largely expressed by mouse SPARC, was transplanted subcutaneously to the mouse. Thus, tumor growth and mouse survival time were examined.

(Results)

The results are shown in the figure. 4. Preventive administration of BM-DC pulsed with SPARC peptide could induce growth inhibition of subcutaneously transplanted METH A and induced prolongation of mouse survival time.

Industrial application

HLA-A24 (A * 2402) is the most frequent HLA class I allele, which is approximately 60% of Japanese. The structural motifs of the peptides bound to the mouse Kd molecules BALB / c are quite similar to those of the peptides bound to the human HLA-A24 molecules. Therefore, it was revealed that, when a BALB / c mouse is immunized with a particular peptide, if the peptide binds to the Kd molecule and thereby produces killing T lymphocytes, and if the killing T lymphocytes destroy the cancer cells expressing a complex of the peptide and the Kd molecule, it is quite possible that the peptide also binds to HLA-A24 and produces human killing T lymphocytes that can destroy cancer cells. Therefore, the peptide of the present invention can be applied to immunotherapy for patients with diffuse infiltrative gastric cancer, pancreatic cancer and melanoma, who possess HLA-A24. It is envisioned that the quality of life of patients can be improved by suppressing the growth or progression of such cancers by cancer immunotherapy based on the SPARC peptide.

Brief description of the drawings

Figure 1 shows the degree of expression of the 15 outstanding genes that were expressed in stomach cancer tissues at a higher level than in the membrane of the normal stomach mucosa, which were identified to perform DNA chip analysis in 20 patients with diffuse infiltrating stomach cancer. Taking into account the results obtained, as well as the results of the normal tissue cDNA chip analysis as described below, from the above 15 genes, cysteine-rich and acid-secreted segregated proteins (SPARC) specifically overexpressed in a Cancer tissue was selected as the most ideal cancer-specific antigen in diffuse infiltrating stomach cancer. Figure 2 shows the results obtained when analyzing the expression of SPARC in 25 types of the main organs of adults and in 4 types of embryonic organs, using a cDNA chip. SPARC gene expression was observed even in some normal tissues, but the level of expression in these normal tissues was significantly lower than in a cancerous tissue. Figure 3 shows the cytotoxicity of SPARC positive tumor cells by CTL that was induced using a SPARC Kd-1, Kd-3 or Kd-4 epitope peptide restricted to mouse Kd in BALB / c mice. The figure. 4 shows the suppression of the growth of Meth A cancer cells transplanted subcutaneously by a cell cancer and the prolongation of the survival time of BALB / c mice by prior administration of charged bone marrow dendrocytes (BM-CC) with a mixture of SPARC peptides Kd-1, Kd-3 and Kd-4.

Sequence listing

<110> University of Kumamoto

<120> Antigenic peptide of rejection of cancer derived from SPARC and pharmaceutical product comprising the same

<130> P2290EP

<140> EP07745372.8

<141> 06-15-2007

<150> JP 20060167724

<151> 06-16-2006

<160> 4

<170> Patentln version 3.3

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<213> Artificial

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<213> Artificial

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Claims (9)

1. A peptide of any of the following:

(TO)

a peptide consisting of the amino acid sequence shown in any of the SEC. ID. No. 1 to No. 3; or

(B)

a peptide consisting of an amino acid sequence comprising a substitution or addition of one

or two amino acids with respect to the peptide consisting of the amino acid sequence shown in any of the SEC. ID. No. 1 to 3, and which has the capacity to produce cytotoxic T lymphocytes (killers); or (C) a peptide consisting of an amino acid sequence comprising an addition of one to 10 amino acids with respect to the peptide consisting of the amino acid sequence shown in any of the SEC. ID. No. 1 to 3, and which has the capacity to produce cytotoxic T lymphocytes (killers).

2.

At least one type of the peptide of claim 1 to produce immunity directed against cancers, for use in the treatment and prevention of tumors.

3.

The peptide of claim 1 for use in the treatment and prevention of tumors.

Four.

At least one type of the peptide of claim 1 for producing antigen presenting cells that has a high capacity to produce T lymphocytes reactive with a tumor, for use in the treatment and prevention of tumors.

5.

At least one type of the peptide of claim 1 for producing T lymphocytes reactive with a tumor, for use in the treatment and prevention of tumors.

6.

A DNA for the induction of antigen presenting cells that has a great capacity to induce T lymphocytes reactive with a tumor, which comprises a gene encoding a peptide of any of the following:

(A), a peptide consisting of the amino acid sequence shown in any of the SEC. ID. No. 1 to 3; or

(B)

a peptide consisting of an amino acid sequence comprising a substitution or addition of one or two amino acids with respect to the peptide consisting of the amino acid sequence shown in any of the SEC. ID. No. 1 to No. 3, and which has the capacity to produce killer T lymphocytes; or

(C)

a peptide consisting of an amino acid sequence comprising an addition of one to 10 amino acids with respect to the peptide consisting of the amino acid sequence shown in any of the SEC. ID. No. 1 to 3, and which has the capacity to produce cytotoxic T lymphocytes (killers).

7.

A killer T lymphocyte, a helper T lymphocyte, or a population of immunocytes comprising said cells, which is produced using the peptide of claim 1.

8.

An antigen presenting cell, which has a complex of an HLA molecule and the peptide of claim 1.

9.

The antigen presenting cell of claim 8, which is pipeted using the peptide of claim 1.